A novel sequence-specific DNA binding protein which interacts with three regularly spaced direct repeats of the CCCTC-motif in the 5'-flanking sequence of the chicken c-myc gene.
The chicken c-myc 5'-flanking sequence has previously been shown to bind multiple proteins present in undifferentiated and differentiated red blood cells. In this report the protein binding to one specific region within a hypersensitive site approximately 200 base pairs upstream of the start of transcription has been analysed in detail. Using a combination of a modified agarose gel retardation assay with O-phenanthroline-copper footprinting in situ, missing contact point and methylation interference techniques, two proteins were found to bind to overlapping sequences within 180-230 bp upstream of the start of transcription. One protein resembles the transcription factor Sp1, the other is a protein which binds to three regularly spaced repeats of the core sequence CCCTC. This CCCTC-binding factor was termed CTCF. It requires additional sequences outside the three recognition motifs for tight binding. CTCF was purified to near homogeneity by sequence-specific DNA chromatography. The approximate molecular weight of the CTCF was estimated to be 130,000. Removal of 110 bp sequence binding both CTCF and Sp1-like proteins leads to a 4 to 8-fold increase in transcription of stably transfected c-myc fusion constructs in chicken embryonic fibroblasts, suggesting that the CTCF is likely to be one of multiple nuclear factors involved in the transcriptional regulation of the chicken c-myc gene.
Lobanenkov
V V
VV
Institute of Carcinogenesis, All-Union Cancer Research Center, Moscow, USSR.
Nicolas
R H
RH
Adler
V V
VV
Paterson
H
H
Klenova
E M
EM
Polotskaja
A V
AV
Goodwin
G H
GH
D000818:Animals; D001482:Base Composition; D001483:Base Sequence; D002642:Chick Embryo; D002846:Chromatography, Affinity; D003596:Cytosine / Q000032:analysis / Q000378:metabolism*; D004247:DNA / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D016259:Genes, myc / Q000235:genetics*; D008745:Methylation; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000235:genetics; D010618:Phenanthrolines; D011401:Promoter Regions, Genetic / Q000235:genetics; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics*; D013941:Thymine / Q000032:analysis / Q000378:metabolism*; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D009841:Oligonucleotides; D010618:Phenanthrolines; D016271:Proto-Oncogene Proteins c-myc; D003596:Cytosine; D004247:DNA; D013941:Thymine; C025205:1,10-phenanthroline